Double-tagged proteins expressed with the His-Strep pQE-TriSystem Vector Set can be isolated by two-step affinity purification to deliver ultrapure (>98% pure) protein (see figure "Ultrapure protein in two steps").

Principle

The His-Strep pQE-TriSystem Vector Set allows expression of proteins with both a 6xHis tag and the Strep-tag II. The double tag allows purification by a two-step affinity purification system, enabling simple and highly efficient purification of ultrapure proteins in a standardized procedure. The system also increases throughput by eliminating the need for development and optimization of protein-specific protocols. Recombinant proteins carrying both tags are purified sequentially on Ni-NTA and Strep-Tactin matrices (see figure "Two-step affinity procedure"). The two simple affinity purifications provide fully active, full-length, and ultrapure protein that is suitable for any downstream application.

Procedure

Recombinant proteins that carry two small affinity tags (the 6xHis tag and Strep-tag II) are efficiently expressed in E. coli, insect, or mammalian cells using pQE-TriSystem His-Strep vectors. After cell lysis and clearing of the lysate, proteins are initially purified using an immobilized-metal affinity chromatography (IMAC) procedure that is based on the proven 6xHis-tag-Ni-NTA interaction. After elution from the Ni-NTA matrix using imidazole, recombinant proteins (which also carry the Strep-tag II epitope) are loaded directly onto a Strep-Tactin matrix (see figure "Two-step affinity purification procedure"). No buffer exchange is required. Protein is eluted from the Strep-Tactin matrix using either biotin or desthiobiotin. Proteins can be detected with high specificity and sensitivity using mouse monoclonal Strep-tag or Anti·His antibodies (see figure "Highly sensitive detection of proteins carrying a Strep-tag").

Applications

The Two-Step Affinity Purification System is highly suited for applications where high purity is essential and is difficult to achieve with His-tag alone, for example, for proteins expressed in eucaryotic cells. The ultrahigh purity and convenience provided by the Two-Step Affinity Purification System make it the method of choice for: